Plant Physiol. (1991) 96, 737-7430032-0889/91/96/0737/07/$01 .00/0

Received for publication November 6, 1990Accepted January 29, 1991

Mechanism of Aluminum Tolerance in Snapbeans'

Root Exudation of Citric Acid

Susan C. Miyasaka*, J. George Buta, Robert K. Howell, and Charles D. FoyUniversity of Hawaii, Hawaii Branch Station, Hilo, Hawaii 96720 (S.C.M.); U.S. Department of Agriculture,

Agricultural Research Service, Plant Hormone Laboratory, BARC-W, Building 050, (J.G.B.) and Climate StressLaboratory, BARC-W, Building 001, Beltsville, Maryland 20705 (R.K.H., C.D.F.)

ABSTRACT

One proposed mechanism of aluminum (Al) tolerance in plantsis the release of an Al-chelating compound into the rhizosphere.In this experiment, two cultivars of snapbeans (Phaseolus vulgarisL. "Romano" and "Dade") that differ in Al tolerance were grownhydroponically with and without Al under aseptic conditions. Aftergrowth in nutrient solutions for 8 days, aliphatic and phenolicorganic acids were analyzed in the culture solutions with an ionchromatograph and a high pressure liquid chromatograph. Thetolerant snapbean, "Dade", when exposed to Al, exuded citricacid into the rhizosphere in a concentration that was 70 times asgreat as that of "Dade" grown without Al, and 10 times as greatas that of "Romano" grown with or without Al. The sensitivecultivar, "Romano", exuded only slightly more citric acid into thegrowing medium under Al-stress, compared to nonstressed con-ditions. Citric acid is known to chelate Al strongly and to reverseits phytotoxic effects. Also, citric acid has been shown previouslyto enhance the availability of phosphorus (P) from insoluble Alphosphates. Thus, one mechanism of Al-tolerance in snapbeansappears to be the exudation of citric acid into the rhizosphere,induced either by toxic levels of Al or by low P due to theprecipitation of insoluble Al phosphates. Our experiment was notable to distinguish between these two factors; however, toleranceto both primary and secondary Al-stress injuries are importantfor plants growing in Al-toxic soils.

Al toxicity is a major factor limiting plant growth in stronglyacid soils (8, 9). Liming is used to correct this problem in theplow layer; however, amendment of acid subsoils is notfeasible economically (9). Moreover, acid soils are found oftenin the tropics and subtropics, where resource-poor farmersare not able to afford such a high-input solution (14).An alternative, low-input solution to this problem is to

utilize the crop plant's genetic potential for tolerance to Alstress (9). Plant species and cultivars within species vary widelyin their resistance to Al injury, and some of these differencesare heritable (9). An increased understanding of the plantmechanisms involved in Al tolerance could help in breedingor selecting plants that are better adapted to acid, Al-toxicsoils.

'Journal Series No. 3518, Hawaii Institute of Tropical Agricultureand Human Resources, University of Hawaii.

One hypothesized mechanism of Al tolerance is the chela-tion and detoxification of Al by organic acids, either withinthe plant (internal tolerance) or in the rhizosphere (exclusion)(8, 9, 30). Organic acids have been reported to chelate Al andto ameliorate its phytotoxic effects (8, 9, 30). Hue et al. (15)showed that the addition of citric, oxalic, and tartaric acids tothe hydroponic solution alleviated the inhibitory effect of Alon root extension of cotton (Gossypium hirsutum L.). Simi-larly, Bartlett and Riego (3) found that Al complexed by citricacid or EDTA did not reduce root and shoot growth of cornplants (Zea mays L.), as did ionic Al. In these studies, theorganic acids could have detoxified Al either externally in therhizosphere or internally after absorption by plant roots.Suhayda and Haug (26-28) found in vitro that organic acids

reversed the Al-induced conformational change in the regu-latory protein, calmodulin, restoring its stimulatory activityon certain enzymes. The following organic acids were able toprotect calmodulin from the deleterious effects of Al in theorder: citric > oxalic > malic > tartaric (26, 27). These resultsdemonstrate that organic acids could protect enzyme activityinternally in the plant from the deleterious effect of Al.

Evidence of internal detoxification by organic acids waspresented by Cambraia et al. (5), who showed that the overallresponse of sorghum (Sorghum bicolor Moench) roots to Alwas an increase in organic acid content. Moreover, the rootsof the Al-tolerant sorghum cultivar contained higher levels oforganic acids, particularly t-aconitic and malic acids, than didthe Al-sensitive cultivar (5). However, in the roots of snap-beans, the overall response to Al was a reduction in organicacid contents, although the Al-tolerant snapbean cultivarmaintained a higher level of organic acids than did the Al-sensitive cultivar (20). Similar results, in which greater Al-tolerance was correlated with higher concentrations oforganicacids in the shoots and roots of plants exposed to Al, werefound for maize (28), barley (Hordeum vulgare L.) (13), andpeas (Pisum sativum L.) (17).A claim for Al exclusion by organic acids was presented by

Ojima and Ohira (23). They showed that a reputedly Al-tolerant carrot (Daucus carota L.) cell line released more citricacid into the suspension culture medium than nonselectedcells (23). Also, they demonstrated that the addition of citricor malic acid into the medium could ameliorate the Al stress(23). However, later evidence showed that the supposedly Al-tolerant cell line was sensitive to ionic Al, because it had been

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selected in the presence of precipitated Al phosphate at a highpH rather than in the presence of ionic Al at a low pH (18).The authors (18) concluded that excretion of organic acids bycarrot cells was a response to low phosphorus (P) availabilityrather than to toxic Al ions.

Certainly, exudation of organic acids into the rhizospherein response to mineral stress has been found for several plants.Alfalfa (Medicago sativa L.) seedlings exuded 182% morecitrate under P stress, than under conditions of sufficient P(21); and citrate has been known to increase P availabilityfrom iron (Fe) and Al phosphates. Also, tea plants (Camelliasinensis [L.] 0. Kuntze), which thrive in acid soils, releasedmalic acid when roots were placed in distilled water (16).However, no evidence has been presented for the release of

organic acids by whole plants into the rhizosphere directly inresponse to Al application. This investigation was conductedto examine the hypothesis that snapbeans exude organic acidsfrom their roots in response to the addition of Al. We soughtto identify these acids, and to compare their quantities in rootexudates of cultivars differing in Al tolerance.

MATERIALS AND METHODS

Two cultivars of snapbeans (Phaseolus vulgaris L., "Ro-mano" and "Dade"), shown previously to differ in Al toler-ance (11), were grown under aseptic conditions, using thefollowing procedures, modified from Barber (1), Barber andGunn (2), Darbyshire and Greaves (7), Kraffczyck et al. (19),and Stotzky et al. (25). The experimental apparatus is illus-trated in Figures 1 and 2.

Snapbeans were grown aseptically, to allow quantificationof the release of organic acids into the rhizosphere, withoutinterference due to microbial metabolism. All supplies weresterilized using an autoclave or ethylene oxide, and all trans-fers were performed in a sterile hood. Seeds were surface-sterilized by soaking in 8.8 M H202 for 15 to 30 min, andthen rinsing in sterilized water. Seedlings were germinated inthe dark for 3 d in Petri dishes, containing nutrient agar

(Difco Laboratories, Detroit, MI)2, and those with no evidenceof bacterial or fungal contamination were grown asepticallyin the apparatus shown in Figure 1. To shield the roots fromexcessive light and heat, the outsides of the centrifuge tubeswere covered by polyethylene sheeting with a white, outerface and a black, inner face. Centrifuge tubes with seedlingswere placed in a battery jar fitted with a glass cover holdingtwo syringe air filters (0.45 gm diameter pores) (Nalge No.199-2045, Nalgene, Rochester, NY). These syringe filterswere sealed to the glass covers, and the glass covers were sealedto the battery jar, using cold-curing silicone rubber (3110RTV, Dow-Coming, Midland, MI). Batteryjars with seedlingswere placed in an environmentally controlled chamber, main-tained at 170 to 200 gmol m-2 s-l photosynthetic photonflux, 28'C day and 220C night, 50% RH, and a 14 hphotoperiod.

After the hypocotyls had elongated (8 d from imbibition),seedlings were selected for vigor and uniformity and placedin the hydroponic system illustrated in Figure 2. Initial at-tempts to seal the plants directly with cold-curing siliconerubber, as suggested by Stotzky et al. (25), adversely affectedthe bean stems. Consequently, polyurethane foam culturetube plugs were cut vertically and used to hold the bean plantsover the nutrient solution and prevent microbial contamina-tion. The polyethylene stoppers containing plants were sealedto the polypropylene jars by silicone rubber and allowed tocure overnight, before aeration was started in the nutrientsolution. Only the roots were maintained under aseptic con-ditions using this system (Fig. 2).

Plants were placed in a growth chamber, maintained at 335to 400 ,Umol m-2 s-' photosynthetic photon flux, 28TC dayand 22°C night, 50% RH, and a 14 h photoperiod. Thenutrient solution was a modified Steinberg solution (10),

'Mention of a product or trade name does not constitute aguarantee or warranty of the product by the U.S. Department ofAgriculture, Agricultural Research Service or the University Hawaii,nor an endorsement over similar products not mentioned.

syringe filters, 0.45 lum pore size

*cold curing silicone rubber

plate glass, with 2 holes drilled, 6 mm diameter

battery jar, glass, 23 cm diameter, 30 cm tall

.snapbeans

- hollow polyethylene stopper. no. 6

Figure 1. Apparatus used to grow snapbeansseedlings aseptically, until the hypocotylelongated.

black polyethylene beads(DFDA-6080-black-4865, Union Carbide, Somerset, NJ)polypropylene centrifuge tube, 100 mL

- polyethylene sheeting, white on outer face, black on inner

- glass beaker

0.6mM CaSO 4 solution

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MECHANISM OF ALUMINUM TOLERANCE IN SNAPBEANS

initially adjusted to pH 4.5. The concentrations of macronu-trients were (in mM): 0.8 K, 1.0 Ca, 0.3 Mg, 0.1 P, 0.3 NH4-N, 3.3 N03-N, and 0.3 S. The concentrations of micronutri-ents were (in gM): 2 Mn, 0.6 B, 0.5 Zn, 0.15 Cu, 0.1 Mo, and10 Fe (as FeEDDHA). Aluminum was added, as AlCl3, at 0and 148 Mm. The Al solution was not autoclaved, but ratherfiltered (0.45 ,um pore diameter), to avoid any precipitationdue to pH changes during autoclaving.The experimental design was a randomized complete block

with five replicates. The treatments were a complete factorialof two cultivars x two Al levels. After 8 d of growth in thesterile nutrient solution (Fig. 2), the plants were harvested andseparated into roots and shoots. All the roots were sonicatedin distilled water for 1 min to remove the loosely adheringsolution from near the root surface, following the method ofBarber and Gunn (2). This sonicated solution was combinedwith the nutrient solution. Shoots and roots were weighed,frozen, freeze-dried, and reweighed. A subsample of the nu-trient solutions (1 mL) was plated onto nutrient agar andchecked for microbial contamination to determine whetheraseptic culture had been maintained throughout theexperiment.A subsample of the nutrient solution (250 mL) was freeze-

dried, redissolved in 10 mL of deionized water, pretreatedwith an On-Guard P cartridge (Dionex Corp., Sunnyvale, CA)to remove phenolics, and run on an ion chromatograph(Dionex Corp., Sunnyvale, CA), using an HPICE-AS5 column(Dionex Corp., Sunnyvale, CA). The eluant was 1.60 mMheptafluorobutyric acid, the regenerant was 5 mm tetrabutyl-ammonium hydroxide, the suppressor was an anion micro-membrane ICE (Dionex Corp., Sunnyvale, CA), and the flowrate was 0.3 mL min-'.

In two replicates, subsamples of the nutrient solutions wererun through an Amberlite XAD-4 resin bed (Sigma ChemicalCo., St. Louis, MO) to trap phenolic compounds. Thesecompounds were removed, with methanol and then analyzedwith reverse phase chromatography (4) on an HPLC.To reconfirm the identity of the organic acid determined

to be citric acid on the ion chromatograph, this peak fromthe Dionex was collected and analyzed on the HPLC, usingthe method of Coppola and Starr (6). All samples were dried

to aeration pump

Table I. Effects of Al on Dry Weights of the Shoots and Roots ofSnapbeans, on Concentrations of Citric Acid in the Growth Medium,and on Final Solution pH

Dry WeightsaCultivar Al Citric Final pH

Shoots Roots

JM 9 AM

Dade 0 0.58 (0.04) 0.20 (0.04) 0.52 (0.33) 6.48 (0.09)Dade 148 0.46 (0.05) 0.13 (0.04) 38.41 (6.97) 4.99 (0.44)Romano 0 0.86 (0.12) 0.35 (0.07) 2.76 (1.20) 6.04 (0.26)Romano 148 0.54 (0.04) 0.16 (0.03) 3.07 (1.93) 4.74 (0.12)

Source P > F: Analysis of Variance

Al 0.0001 0.0001 0.0001 0.0001Cultivar 0.0001 0.0010 0.0001 0.2160Cult x Al 0.0090 0.0110 0.0001 0.7340a Means are followed by standard deviations in parentheses.

in a rotary evaporator, redissolved in methanol, and runthrough two PLRP-S columns (Polymer Lab., Amherst, MA)in series, using a mobile phase of 0.2 M KH2PO4 buffer (pH2.4), with 0.2 M acetonitrile (6).

Analysis of variance (ANOVA) was calculated, using theSAS computer program (24). A probability level of 0.05 orless was considered statistically significant.

RESULTS AND DISCUSSION

Aseptic Culture

Cultures of the nutrient solutions showed that only 2 outof 20 containers had microbial contamination, indicating thesuccess of the aseptic culture system (Figs. 1, 2). Withoutaseptic culture, quantification of organic acids in the nutrientmedium would be suspect, because of microbial breakdownof organic acids.

Dry Weights

Aluminum at 148 ,M significantly reduced the dry weightsof the shoots and roots of both snapbean cultivars (Table I).

- snapbean

- culture tube plug. foamed polyurethane, diameter 35 mm

syringe filters, 0.45 gm pore size

- cold-curing silicone rubber

- hollow polyethylene stopper, no. 6

' black polyethylene beads

(DFDA-6080-black-4865, Union Carbide, Somerset, NJ)

- teflon tubing. O.D. 4 mm, I.D. 2.5 mm

Figure 2. Hydroponic system used to growsnapbean seedlings with roots maintained in anaseptic environment.

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(A0

4-0)

0._

I=

0 10 20 30 40Time, minutes

Figure 3. Elution peaks on an ion chromatograph of the followingstandard organic acids: malonic, malic, acetic, citric, and succinic.Chromatograms of the organic acids are shown in the presence andabsence of Al.

Also, significant cultivar differences were found, as well assignificant interaction between cultivar and Al treatments(Table I). This interaction was due to the greater Al-induceddecrease in dry weights of the shoots and roots of the Al-sensitive cultivar, "Romano", compared with those of the Al-tolerant cultivar, "Dade" (Table I).Although dry weights of the roots of both cultivars exposed

to 148 gM Al appeared similar (Table I), this comparison wasmisleading. The root tips of the Al-sensitive cultivar "Ro-mano" died when exposed to Al, resulting in a mass of rootsin the moist air above the solution. In contrast, the primaryroots of the Al-tolerant cultivar "Dade" survived and grew inthe solutions containing Al, although the secondary rootswere stunted.

Solution pH

Solution pH was initially adjusted to 4.5, and then notcontrolled during the experiment. Final solution pH levelsare reported in Table I. The nutrient solution contained bothNH4' and NO3-, which resulted in an initial decline in pH,followed by a subsequent increase in pH as plants grew (10,11). Aluminum at 148 uM resulted in significantly lower finalsolution pH levels compared to treatments with no Al (TableI), probably as a result of depressed shoot and root growth in

the presence of Al. No significant cultivar effect was foundfor final solution pH (Table I), supporting the earlier resultsof Foy et al. (1 1), in which differential Al tolerance of snap-beans was not found to be associated with differential solutionpH changes.

Aliphatic Organic Acids

Sonication of roots in distilled water for 1 min was used toremove loosely adhering soution from near the root surface,along with organic acids external to the roots. Barber andGunn (2) used sonication for 5 min to remove organic sub-stances from the outside of cereal roots, and found that thestructure of root hairs, protoplasmic streaming, and subse-quent uptake of phosphate by cereal roots were not signifi-cantly affected by this treatment.

Representative traces from the ion chromatograph areshown in Figures 3, 4, and 5. The addition of Al to thestandard organic acids resulted in an earlier elution time forcitric acid, as well as a broadening of the peak (Fig. 3),indicating chelation between Al and citric acid. Although thepeak heights were different, the areas under the curve weresimilar for citric acid with or without Al.

In the absence of Al, the Al-sensitive "Romano" seedlingsexuded malic, citric, and an unknown acid that eluted at 19.4min (Fig. 4). In the presence of 148 ,uM Al, "Romano"seedlings released citric acid at a slightly higher concentrationthan in the absence of Al (Fig. 4; Table I). The large peak

CD

ao C f|_Romano, - Al

or148AM Ml.

C~~~~~~~~~C

0 10 20 30 40Time, minutes

Figure 4. Elution peaks on an ion chromatograph of organic acidsexuded by "Romano" seedlings into nutrient solutions, containing 0or 148 Am Al.

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MECHANISM OF ALUMINUM TOLERANCE IN SNAPBEANS

C~~~~~~~~~~~~CC~~~~~~~~~~~~C

0 10 20 30 40

Time, minutes

Figure 5. Elution peaks on an ion chromatograph of organic acidsexuded by "Dade" seedlings into nutrient solutions, containing 0 or148 AM Al.

preceding that of citric acid was due to the high concentrationsof inorganic ions (i.e. K+, Ca2+, Mg2+, NH4+, and NO3-) thatwere not absorbed by the Al-stressed plants (Fig. 4). This largepeak possibly obscured malic acid and other organic acidsthat eluted prior to citric acid.

In the absence of Al, the Al-tolerant "Dade" exuded malic,and small amounts of citric acid, as well as the unknown acidthat eluted at 19.4 min (Fig. 5; Table I). In the presence of148 Mm Al, "Dade" exuded 70 times as much citric acid as inthe absence of Al, and 10 times as much citric acid as"Romano" grown with or without Al (Figs. 4, 5; Table I).Again, the large peak prior to that of citric acid was due toinorganic ions, which might have obscured the elution ofother acids, such as malic. To our knowledge, this is the firstreport that the roots of an Al-tolerant plant cultivar exudedcitric acid into the growth medium, directly in response tothe addition of Al.

Citric acid has been reported to chelate and detoxify Al (3,15, 23, 28). Certainly, in this experiment, the primary rootsof the Al-tolerant cultivar "Dade" were able to continuegrowing in solutions containing Al, whereas the roots of theAl-sensitive cultivar "Romano" died as soon as they contactedthose solutions. Apparently the release of a large concentra-tion of citric acid into the growing medium (Table I) allowedthe roots of the tolerant cultivar to survive and grow innutrient solutions with 148 Mm Al.

Similar results were found in a preliminary experiment, inwhich exposure to 74 ALM Al resulted in the release of citricacid by Al-tolerant "Dade" at a concentration 10 times greaterthan that released in the absence of Al, and 100 times greaterthan that released by Al-sensitive "Romano" in the presenceof Al (data not shown). No detectable amount of citric acidwas found in the rhizosphere of "Romano" grown in theabsence of Al. At 74 AM Al, the roots of "Romano" wereslightly stunted, with only one out of five plants exhibitingthe "coralloid" roots typical of Al toxicity. The roots of Al-tolerant "Dade", at this low level of Al stress, appearedhealthy, with only two out of five plants showing someinhibition of secondary roots.

Results from our experiments provide evidence for thehypothesis that one mechanism of Al exclusion in snapbeansis the release of citric acid into the growth medium containingAl. Taylor (30) questioned the exudation of chelates as amechanism of tolerance due to the considerable energeticcost. Our experiments showed that the release of a highconcentration of citric acid by the Al-tolerant cultivar wasinduced by the addition of Al, thereby limiting the metaboliccost to a situation where continued growth of the root systemwas dependent on amelioration of Al effects. Also, the ener-getic cost to the plant would be kept to a minimum, if citricacid in the rhizosphere is needed to protect primarily the rootapex, rather than the entire length of the root.These results support the hypothesis of Lee and Foy (20)

that organic acids are involved in differential Al tolerance ofsnapbeans; however, the mechanism appears to occur exter-nally rather than internally. The exudation of organic acidsinto the rhizosphere in response to Al could explain the overalldecrease in organic acid levels ofsnapbeans found by Lee andFoy (20). Also, an external release of organic acids mightexplain the lack of correlations found between Al toleranceand changes in internal organic acid contents of wheat ( 12).

Recent work by Koyama et al. (18) raised the question asto whether the exudation of citric acid by Al-tolerant "Dade"was in response to toxic Al ions or in response to low P

. 0.00

0.0 .10-0n8r

0 10 15Time, minutes

20

Figure 6. Elution peaks on an HPLC of the following organic acids:malic, malonic, citric, and succinic. Also, the elution peaks on anHPLC of the sample collected from the ion chromatograph followingthe citric acid peak.

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availability due to the precipitation of Al phosphate. In ourexperiment, it was not possible to separate the effects of toxicAl stress from low P stress, because the addition ofAl probablyresults in the precipitation ofsome aluminum phosphate evenat pH 4.5. However, induced phosphate deficiency is consid-ered to be part of the total Al toxicity syndrome (29). Also,Al tolerance has been reported to be associated with the abilityto tolerate low P levels in solution (9, 29). Earlier work byFoy et al. ( 1) showed that the addition of 148 jAM Al hadlittle or no effect on P concentrations in the stem exudates ofAl-tolerant "Dade", but resulted in greatly reduced P concen-trations in those of Al-sensitive "Romano." Perhaps, part ofthe mechanism of Al tolerance in "Dade" is due to its abilityto maintain uptake of P through the release of citric acid intothe rhizosphere and the subsequent solubilization of P fromAl phosphates.

In wheat, differential Al tolerance was associated with dif-ferences in ion transport across membranes (22). Perhaps, insnapbeans, chelation of Al in the rhizosphere by citric acidcould serve to protect membrane functions from the delete-rious effects of Al. Alternatively, solubilization of precipitatedAl phosphate by citric acid could help to increase uptake ofP, and thus maintain other transport functions dependent onP. It would be interesting to measure ion transport andmembrane potentials of these two snapbean cultivars, andrelate the exudation of citric acid to these measurements.Perhaps, then, these microtechniques could be used to deter-mine whether the release of organic acids is localized at theroot apex, where its protective effect might be most critical.More than one mechanism could be responsible for Al-

tolerance in snapbeans. Certainly, the roots of both snapbeancultivars were able to grow in a medium containing 74 gmAl, despite the fact that only "Dade" released citric acid intothe rhizosphere. Further research needs to be carried out toexamine other possible mechanisms of Al tolerance in snap-beans and to determine whether the release of citric acid byan Al-tolerant cultivar is a direct response to Al stress or asecondary response to low P due to precipitation of Alphosphate.

Phenolic Organic Acids

Analyses for phenolic acids did not reveal any consistentpattern of phenolic compounds released into the rhizosphereof snapbean cultivars, grown with or without Al (data notshown). It is therefore likely that production of phenolic acidsis not a major mechanism involved in Al tolerance ofsnapbeans.

Citric Acid

To reconfirm the identification of the organic acid releasedin large quantities by Al-tolerant "Dade" snapbeans in thepresence of Al, the peak was collected from the ion chromat-ograph, and run through the HPLC. The citric acid peakcollected from the ion chromatograph eluted earlier than thestandard citric acid (Fig. 6); however, results showed that thepresence of Al resulted in an earlier elution time of citric acid(data not shown). Thus, the results from the HPLC supportthe identification of citric acid as the organic acid released bythe Al-tolerant cultivar in response to the addition of Al.

CONCLUSIONS

One proposed mechanism of Al tolerance is the release ofchelating agents, such as organic acids, into the rhizosphere.In our experiment, the roots ofAl-tolerant snapbeans exposedto 148 gM Al exuded 70 times as much citric acid as in theabsence of Al, and 10 times as much citric acid as "Romano"grown with or without Al. Citric acid has been reportedpreviously to chelate Al and reverse its phytotoxic effects.Also, release of citric acid has been known to enhance avail-ability of P from insoluble sources, such as Al phosphate.Evidence from our experiment is the first report ofAl-tolerantwhole plants releasing citric acid into the rhizosphere inresponse to the addition of Al. Our experiment was not ableto distinguish between response to toxic Al stress or to low Pstress. However, tolerance to either direct Al-stress injuries orto secondary ones is important for plants growing in Al-toxicsoils.

ACKNOWLEDGMENTS

The authors thank R. Mirecki for his assistance with the ionchromatograph and the environmental chamber, and D. T. Ida andH. Mishima for their assistance with the scientific illustrations. Also,they acknowledge the generous donation of "Dade" and "Romano"snapbean seeds by Sun Seeds (Hollister, CA).

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